Human sodium-dependent phosphate cotransporter

ABSTRACT

The present invention provides a human sodium-dependent phosphate cotransporter (NAPTR) and polynucleotides which identify and encode NAPTR. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding NAPTR and a method for producing NAPTR. The invention also provides for agonists, antibodies, or antagonists specifically for NAPTR. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding NAPTR for the treatment of diseases associated with the expression of NAPTR. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding NAPTR. The invention also provides a method for treating disorders associated with decreased phosphate levels by administering NAPTR and a method for treating disorders associated with increased phosphate levels by administering antagonists to NAPTR.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of anovel human sodium-dependent phosphate cotransporter and to the use ofthese sequences in the diagnosis, prevention, and treatment of diseasesassociated with increased or decreased phosphate levels.

BACKGROUND OF THE INVENTION

Phosphate is present in the plasma, intracellular fluid, cell membranes,collagen and bone tissue of mammals. It is a dynamic constituent ofenergy metabolism, an essential component of skeletal mineralization, amodulator of tissue concentrations of calcium, and plays a major role inrenal excretion of H⁺.

Phosphate homeostasis in mammals is a balance between intake, intestinalabsorption, bone depositionlresorption, and renal excretion andresorption. An excess of phosphate reduces the circulating Ca²⁺ levels,and a deficit results in decreases in erythrocyte ATP and2,3-diphosphoglycerate and contributes to the pathology of osteomalacia,hypocalciuria, and rickets. Dietary phosphate is absorbed from thegastrointestinal tract in an active, energy-dependent process that ismodified by hormones, vitamin D, Ca²⁺, and Al³⁺. Regulation of the serumconcentration of phosphate is maintained through resorption by thesodium phosphate cotransport system, located in the proximal convolutedrenal tubule. Local concentration of phosphate in specific tissue types,such as liver, bone, and brain, is modulated by sodium phosphatetransport proteins located in these tissues. (Hartmann, C. et al. (1996)Proc. Natl. Acad. Sci. 93:7409-7414; Glinn, M. et al.(1995) J.Neurochem. 65:2358-2365).

Human NPT 1, NPT2, NaP₁ -3, and the X-linked hypophosphatemia (PEX)sodium phosphate transport proteins are found in the renal brush bordermembrane where they participate in renal tubular phosphate uptake.Although similar in function, these renal proteins differ in affinity,capacity, map to different chromosomal locations, and are differentiallyregulated by hormones and dietary phosphate (Tenenhouse, H. (1989)Biochem. Biophys. Acta 984: 207-213; Fulceri, R. (1993) Biochem. J.289:299-306; Chong, S. et al. (1993) Genomics 18:355-359; Miyamoto, K.et al. (1995) Biochem. J. 305:81-85).

Sodium phosphate transport proteins in rat brain neurons regulateintracellular phosphate concentrations necessary for maintaining thephosphorylation potential of the cell. Physiological concentrations ofphosphate enhance the ATP-dependent binding of Ca²⁺ to brain microsomes,resulting in a larger intracellular pool of Ca²⁺ released by inositoltriphosphate. The expression of the brain specific sodium-dependentphosphate transporter, rBNPI, is developmentally regulated and isspecific to neuron enriched regions of the adult rat brain. Avianosteoclasts express a sodium-dependent phosphate transporter regulatedthrough integrin-mediated pathways in the presence of bone. Thistransporter is hypothesized to act in the transcellular movement ofphosphate during active bone resorption (Ni, B. (1995) J. Neurosci. 15:5789-5799; Gupta, A. (1996) Kidney Int. 46: 968-974).

By low stringency screening of a human kidney cortex cDNA library with arabbit NaP1-1 cDNA, Chong et.al. (1993, supra) isolated a cDNA encodinga human sodium-dependent phosphate transport protein (NPT1).Localization of NPT1 to 6p23-p21.3 was found by Southern hybridizationto HindIII-digested DNA from a human chromosome 6 somatic cell hybriddeletion panel. Fluorescence in situ hybridization maps NPT1 to 12p11 inthe rabbit. This assignment agrees with the previously reported homologybetween rabbit chromosome 12 and human chromosome 6 (Kos, C. et al.(1994) Genomics 19: 176-177).

The discovery of proteins related to human renal sodium phosphatetransport protein, and the polynucleotides encoding them, satisfies aneed in the art by providing new compositions useful in diagnosis andtreatment of diseases associated with increased or decreased phosphatelevels.

SUMMARY OF THE INVENTION

The present invention features a novel human sodium-dependent phosphatecotransporter hereinafter designated NAPTR and characterized as havingsimilarity to human renal sodium phosphate transport protein and ratbrain-specific sodium-dependent inorganic phosphate cotransporter.

Accordingly, the invention features a substantially purified NAPTRhaving the amino acid sequence shown in SEQ ID NO:1.

One aspect of the invention features isolated and substantially purifiedpolynucleotides that encode NAPTR. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2.

The invention also relates to a polynucleotide sequence comprising thecomplement of SEQ ID NO:2 or variants thereof. In addition, theinvention features polynucleotide sequences which hybridize understringent conditions to SEQ ID NO:2.

The invention additionally features nucleic acid sequences encodingpolypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments,portions or antisense molecules thereof, and expression vectors and hostcells comprising polynucleotides that encode NAPTR. The presentinvention also features antibodies which bind specifically to NAPTR, andpharmaceutical compositions comprising substantially purified NAPTR. Theinvention also features agonists and antagonists of NAPTR. The inventionalso features a method for treating disorders associated with decreasedphosphate levels by administering NAPTR and a method for treatingdisorders associated with increased phosphate levels by administering anantagonist to NAPTR

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of NAPTR. The alignment was producedusing MacDNASIS PRO™ software (Hitachi Software Engineering Co., Ltd.,San Bruno, Calif.).

FIGS. 2A and 2B show the amino acid sequence alignments among NAPTR (SEQID NO:1), human sodium phosphate transporter protein (GI 450532; SEQ IDNO:3), and rat rain-specific sodium-dependent inorganic phosphatecotransporter (GI 507415; SEQ ID NO:4). The alignment was produced usingthe multisequence alignment program of DNASTAR™ software (DNASTAR Inc,Madison Wis.).

FIGS. 3A, 3B, and 3C show the hydrophobicity plots (MacDNASIS PROsoftware) for NAPTR, SEQ ID NO: 1, human sodium phosphate transporterprotein, SEQ ID NO:3, and rat brain-specific sodium-dependent inorganicphosphate cotransporter, SEQ ID NO:4, respectively. The positive X axisreflects amino acid position, and the negative Y axis, hydrophobicity.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

DEFINITIONS

"Nucleic acid sequence" as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand. Similarly,"amino acid sequence" as used herein refers to an oligopeptide, peptide,polypeptide, or protein sequence, and fragments or portions thereof, andto naturally occurring or synthetic molecules.

Where "amino acid sequence" is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, "amino acidsequence" and like terms, such as "polypeptide" or "protein" are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

"Peptide nucleic acid", as used herein, refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

NAPTR, as used herein, refers to the amino acid sequences ofsubstantially purified NAPTR obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

"Consensus", as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, or which has been extendedusing XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3'direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GELVIEW™Fragment Assembly system (GCG, Madison, Wis.), or which has been bothextended and assembled.

A "variant" of NAPTR, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

A "deletion", as used herein, refers to a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent.

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid or nucleotide residues, respectively, as compared to thenaturally occurring molecule.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic NAPTR, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The term "agonist", as used herein, refers to a molecule which, whenbound to NAPTR, causes a change in NAPTR which modulates the activity ofNAPTR. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to NAPTR.

The terms "antagonist" or "inhibitor", as used herein, refer to amolecule which, when bound to NAPTR, blocks or modulates the biologicalor immunological activity of NAPTR. Antagonists and inhibitors mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to NAPTR.

The term "modulate", as used herein, refers to a change or an alterationin the biological activity of NAPTR. Modulation may be an increase or adecrease in protein activity, a change in binding characteristics, orany other change in the biological, functional or immunologicalproperties of NAPTR.

The term "mimetic", as used herein, refers to a molecule, the structureof which is developed from knowledge of the structure of NAPTR orportions thereof and, as such, is able to effect some or all of theactions of NAPTR-like molecules.

The term "derivative", as used herein, refers to the chemicalmodification of a nucleic acid encoding NAPTR or the encoded NAPTR.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

"Amplification" as used herein refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term "hybridization", as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen binds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides by base-pairing. For example, forthe sequence "A-G-T" binds to the complementary sequence "T-C-A".Complementarity between two single-stranded molecules may be "partial",in which only some of the nucleic acids bind, or it may be complete whentotal complementarity exists between the single stranded molecules. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands.

The term "homology", as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term"substantially homologous." The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

As known in the art, numerous equivalent conditions may be employed tocomprise either low or high stringency conditions. Factors such as thelength and nature (DNA, RNA, base composition) of the sequence, natureof the target (DNA, RNA, base composition, presence in solution orimmobilization, etc.), and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfateand/or polyethylene glycol) are considered and the hybridizationsolution may be varied to generate conditions of either low or highstringency different from, but equivalent to, the above listedconditions.

The term "stringent conditions", as used herein, is the "stringency"which occurs within a range from about Tm-5° C. (5° C. below the meltingtemperature (Tm) of the probe) to about 20° C. to 25° C. below Tm. Aswill be understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences.

The term "antisense", as used herein, refers to nucleotide sequenceswhich are complementary to a specific DNA or RNA sequence. The term"antisense strand" is used in reference to a nucleic acid strand that iscomplementary to the "sense" strand. Antisense molecules may be producedby any method, including synthesis by ligating the gene(s) of interestin a reverse orientation to a viral promoter which permits the synthesisof a complementary strand. Once introduced into a cell, this transcribedstrand combines with natural sequences produced by the cell to formduplexes. These duplexes then block either the further transcription ortranslation. In this manner, mutant phenotypes may be generated. Thedesignation "negative" is sometimes used in reference to the antisensestrand, and "positive" is sometimes used in reference to the sensestrand.

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein "comprising atleast a portion of the amino acid sequence of SEQ ID NO:1" encompassesthe full-length human NAPTR and fragments thereof.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

The term "antigenic determinant", as used herein, refers to that portionof a molecule that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The terms "specific binding" or "specifically binding", as used herein,in reference to the interaction of an antibody and a protein or peptide,mean that the interaction is dependent upon the presence of a particularstructure (i.e., the antigenic determinant or epitope) on the protein;in other words, the antibody is recognizing and binding to a specificprotein structure rather than to proteins in general. For example, if anantibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding NAPTR orfragments thereof may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2 by northern analysis is indicative of thepresence of mRNA encoding NAPTR in a sample and thereby correlates withexpression of the transcript from the polynucleotide encoding theprotein.

"Alterations" in the polynucleotide of SEQ ID NO: 2, as used herein,comprise any alteration in the sequence of polynucleotides encodingNAPTR including deletions, insertions, and point mutations that may bedetected using hybridization assays. Included within this definition isthe detection of alterations to the genomic DNA sequence which encodesNAPTR (e.g., by alterations in the pattern of restriction fragmentlength polymorphisms capable of hybridizing to SEQ ID NO:2), theinability of a selected fragment of SEQ ID NO: 2 to hybridize to asample of genomic DNA (e.g., using allele-specific oligonucleotideprobes), and improper or unexpected hybridization, such as hybridizationto a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding NAPTR (e.g., using fluorescent in situhybridization [FISH] to metaphase chromosomes spreads).

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fa, F(ab')₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind NAPTRpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

THE INVENTION

The invention is based on the discovery of a novel humansodium-dependent phosphate cotransporter, NAPTR, the polynucleotidesencoding NAPTR, and the use of these compositions for the diagnosis,prevention, or treatment of disorders associated with increased ordecreased phosphate levels.

Nucleic acids encoding the human NAPTR of the present invention werefirst identified in Incyte Clone 754412 from the brain tumor cDNAlibrary (BRAITUT02) through a computer-generated search for amino acidsequence alignments. A consensus sequence, SEQ ID NO:2, was derived fromthe extension of the nucleic acid sequence of Incyte Clone 754412.est(BRAITUT02).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIG. 1. NAPTR is 402amino acids in length and has chemical and structural homology withhuman renal sodium phosphate transport protein (GI 450532; SEQ ID NO:3)and rat brain-specific sodium-dependent inorganic phosphatecotransporter (GI 507415; SEQ ID NO:4). In particular, NAPTR, humanrenal sodium phosphate transport protein, and rat brain-specificsodium-dependent inorganic phosphate cotransporter share 48% and 29%identity, respectively and all three have a potential N-glycosylationsites at N₄₉, N₄₉, and N₉₂, respectively. As illustrated by FIGS. 3A,3B, and 3C, NAPTR, human renal sodium phosphate transport protein, andrat brain-specific sodium-dependent inorganic phosphate cotransporterhave rather similar hydrophobicity plots.

The invention also encompasses NAPTR variants. A preferred NAPTR variantis one having at least 80%, and more preferably 90%, amino acid sequenceidentity to the NAPTR amino acid sequence (SEQ ID NO:1). A mostpreferred NAPTR variant is one having at least 95% amino acid sequenceidentity to SEQ ID NO:1.

The invention also encompasses polynucleotides which encode NAPTR.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of NAPTR can be used to generate recombinant molecules whichexpress NAPTR. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIG. 1.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding NAPTR, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring NAPTR, and all such variations are to beconsidered as being specifically disclosed.

Although nucleotide sequences which encode NAPTR and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring NAPTR under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding NAPTR or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding NAPTR and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or portionsthereof, which encode NAPTR and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art at the time of the filing ofthis application. Moreover, synthetic chemistry may be used to introducemutations into a sequence encoding NAPTR or any portion thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

Altered nucleic acid sequences encoding NAPTR which are encompassed bythe invention include deletions, insertions, or substitutions ofdifferent nucleotides resulting in a polynucleotide that encodes thesame or a functionally equivalent NAPTR. The encoded protein may alsocontain deletions, insertions, or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentNAPTR. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of NAPTR is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyrosine.

Also included within the scope of the present invention are alleles ofthe genes encoding NAPTR. As used herein, an "allele" or "allelicsequence" is an alternative form of the gene which may result from atleast one mutation in the nucleic acid sequence. Alleles may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given gene may have none, one, or many allelic forms.Common mutational changes which give rise to alleles are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any embodiments of the invention. Themethods may employ such enzymes as the Klenow fragment of DNA polymeraseI, Sequenase® (US Biochemical Corp, Cleveland, Ohio), Taq polymerase(Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, Ill.), orcombinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE Amplification System marketed by Gibco BRL(Gaithersburg, Md.). Preferably, the process is automated with machinessuch as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), PeltierThermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI 377DNA sequencers (Perkin Elmer).

The nucleic acid sequences encoding NAPTR may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,"restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 Primer Analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PromoterFinder™libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5' and 3'non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devisecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. Genotyper™ and Sequence Navigator™,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode NAPTR, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of NAPTR in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressNAPTR.

As will be understood by those of skill in the art, it may beadvantageous to produce NAPTR-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter NAPTRencoding sequences for a variety of reasons, including but not limitedto, alterations which modify the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding NAPTR may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of NAPTR activity, it may be useful toencode a chimeric NAPTR protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the NAPTR encoding sequence and theheterologous protein sequence, so that NAPTR may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding NAPTR may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of NAPTR, or a portion thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of NAPTR, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active NAPTR, the nucleotidesequences encoding NAPTR or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding NAPTR andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding NAPTR. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector--enhancers, promoters, 5' and 3'untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript® phagemid (Stratagene,LaJolla, Calif.) or pSport1™ plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding NAPTR,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for NAPTR. For example, when largequantities of NAPTR are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBluescript®) (Stratagene), in which the sequence encoding NAPTR may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of 13-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding NAPTR may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196.

An insect system may also be used to express NAPTR. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding NAPTR may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of NAPTR will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which NAPTR may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding NAPTR may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing NAPTR in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding NAPTR. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding NAPTR, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressNAPTR may be transformed using expression vectors which may containviral origins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransfcrase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-13 1).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding NAPTR isinserted within a marker gene sequence, recombinant cells containingsequences encoding NAPTR can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding NAPTR under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding NAPTR and express NAPTR may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA--DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding NAPTR can be detectedby DNA--DNA or DNA-RNA hybridization or amplification using probes orportions or fragments of polynucleotides encoding NAPTR. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding NAPTR to detect transformantscontaining DNA or RNA encoding NAPTR. As used herein "oligonucleotides"or "oligomers" refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nuclcotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides, which canbe used as a probe or amplimer.

A variety of protocols for detecting and measuring the expression ofNAPTR, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson NAPTR is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding NAPTR includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding NAPTR, orany portions thereof may be cloned into a vector for the production ofan mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland, Ohio).Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with nucleotide sequences encoding NAPTR may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeNAPTR may be designed to contain signal sequences which direct secretionof NAPTR through a prokaryotic or eukaryotic cell membrane. Otherrecombinant constructions may be used to join sequences encoding NAPTRto nucleotide sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and NAPTR may be used to facilitate purification.One such expression vector provides for expression of a fusion proteincontaining NAPTR and a nucleic acid encoding 6 histidine residuespreceding a thioredoxin or an enterokinase cleavage site. The histidineresidues facilitate purification on IMIAC (immobilized metal ionaffinity chromatography as described in Porath, J. et al. (1992, Prot.Exp. Purif. 3: 263-281) while the enterokinase cleavage site provides ameans for purifying NAPTR from the fusion protein. A discussion ofvectors which contain fusion proteins is provided in Kroll, D. J. et al.(1993; DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of NAPTR may beproduced by direct peptide synthesis using solid-phase techniquesMerrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431APeptide Synthesizer (Perkin Elmer). Various fragments of NAPTR may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

THERAPEUTICS

Based on the chemical and structural homology between NAPTR and humanNPT1, NAPTR is a sodium-dependent phosphate cotransporter and appears toplay a role in the regulation of phosphate levels. Increases ordecreases of the level of phosphate in a subject that are above or belownormal physiological values are a cause of harm to the subject.

Therefore, in one embodiment, NAPTR or a fragment or derivative thereofmay be administered to a subject to treat or prevent disordersassociated with decreased phosphate levels. Such disorders may include,but are not limited to cancers of the kidney, disorders of decreasedphosphate levels including tumoral calcinosis, osteomalacia,osteoporosis, familial hypophosphatemia, rickets, cysteneuria,nephrocalcinosis, glomerulonephritis, renal calculus, Alzheimersdisease, diabetes melitis, hereditary amyloidosis, myopathies includingprogressive external ophthalmoplegia, Kearns-Sayre syndrome, myoclonicepilepsy, encephalopathy, and cardiomyopathy, hypokalemia,Goodpastures'Syndrome, and disorders of cell signaling through cAMP,ATP, NADPH, and glucose-6-phosphate.

In another embodiment, a vector capable of expressing NAPTR, or afragment or a derivative thereof, may also be administered to a subjectto treat the disorders associated with decreased phosphate levels listedabove.

In another embodiment, antagonists or inhibitors of NAPTR may beadministered to a subject to treat or prevent disorders associated withincreased phosphate levels. Such disorders may include, but are notlimited to, disorders of increased phosphate levels including,hypocalciuria, hypocalcemia, and abnormal phosphate regulation inneurons, gastrointestinal tract, and liver. In one aspect, antibodieswhich are specific for NAPTR may be used directly as an antagonist, orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express NAPTR. Antibodieswhich are specific for NAPTR may be used directly as an antagonist, orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express NAPTR.

In another embodiment, a vector expressing antisense of thepolynucleotide encoding NAPTR may be administered to a subject to treator prevent the disorders associated with increased phosphate levelslisted above.

In other embodiments, any of the therapeutic proteins, antagonists,antibodies, agonists, antisense sequences or vectors described above maybe administered in combination with other appropriate therapeuticagents. Selection of the appropriate agents for use in combinationtherapy may be made by one of ordinary skill in the art, according toconventional pharmaceutical principles. The combination of therapeuticagents may act synergistically to effect the treatment or prevention ofthe various disorders described above. Using this approach, one may beable to achieve therapeutic efficacy with lower dosages of each agent,thus reducing the potential for adverse side effects.

Antagonists or inhibitors of NAPTR may be produced using methods whichare generally known in the art. In particular, purified NAPTR may beused to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind NAPTR.

Antibodies specific to NAPTR may be generated using methods that arewell known in the art. Such antibodies may include, but are not limitedto, polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith NAPTR or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the peptides, fragments, or oligopeptides used toinduce antibodies to NAPTR have an amino acid sequence consisting of atleast five amino acids, and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of NAPTR amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

Monoclonal antibodies to NAPTR may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.62:109-120).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceNAPTR-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for NAPTR mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between NAPTR and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering NAPTR epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingNAPTR, or any fragment thereof, or antisense molecules, may be used fortherapeutic purposes. In one aspect, antisense to the polynucleotideencoding NAPTR may be used in situations in which it would be desirableto block the transcription of the mRNA. In particular, cells may betransformed with sequences complementary to polynucleotides encodingNAPTR. Thus, antisense molecules may be used to modulate NAPTR activity,or to achieve regulation of gene function. Such technology is now wellknown in the art, and sense or antisense oligomers or larger fragments,can be designed from various locations along the coding or controlregions of sequences encoding NAPTR.

Expression vectors derived from retro viruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encodingNAPTR. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

Genes encoding NAPTR can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes NAPTR. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA, or PNA, to the control regionsof the gene encoding NAPTR, i.e., the promoters, enhancers, and introns.Oligonucleotides derived from the transcription initiation site, e.g.,between positions -10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using "triple helix" base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The antisense molecules may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding NAPTR.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of nucleic acid molecules.These include techniques for chemically synthesizing oligonucleotidessuch as solid phase phosphoramidite chemical synthesis. Alternatively,RNA molecules may be generated by in vitro and in vivo transcription ofDNA sequences encoding NAPTR. Such DNA sequences may be incorporatedinto a wide variety of vectors with suitable RNA polymerase promoterssuch as T7 or SP6. Alternatively, these cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of NAPTR, antibodies toNAPTR, mimetics, agonists, antagonists, or inhibitors of NAPTR. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of NAPTR, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example NAPTR or fragments thereof, antibodies of NAPTR,agonists, antagonists or inhibitors of NAPTR, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind NAPTR may beused for the diagnosis of conditions or diseases characterized byexpression of NAPTR, or in assays to monitor patients being treated withNAPTR, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for NAPTR includemethods which utilize the antibody and a label to detect NAPTR in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuringNAPTR are known in the art and provide a basis for diagnosing altered orabnormal levels of NAPTR expression. Normal or standard values for NAPTRexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toNAPTR under conditions suitable for complex formation The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric, means. Quantities of NAPTR expressed insubject, control and disease, samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingNAPTR may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, antisense RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofNAPTR may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression ofNAPTR, and to monitor regulation of NAPTR levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding NAPTR or closely related molecules, may be used to identifynucleic acid sequences which encode NAPTR. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding NAPTR, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe NAPTR encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring NAPTR.

Means for producing specific hybridization probes for DNAs encodingNAPTR include the cloning of nucleic acid sequences encoding NAPTR orNAPTR derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding NAPTR may be used for the diagnosis ofdisorders which are associated with expression of NAPTR. Examples ofsuch disorders associated with decreased expression of NAPTR includecancers of the kidney, disorders of decreased phosphate levels includingtumoral calcinosis, osteomalacia, osteoporosis, familialhypophosphatemia, rickets, cysteneuria, nephrocalcinosis,glomerulonephritis, renal calculus, Alzheimers disease, diabetesmellitus, hereditary amyloidosis, myopathies including progressiveexternal ophthalmoplegia, Kearns-Sayre syndrome, myoclonic epilepsy,encephalopathy, and cardiomyopathy, hypokalemia, Goodpastures' Syndrome,and disorders of cell signaling through cAMP, ATP, NADPH, andglucose-6-phosphate. Examples of disorders associated with increasedexpression of NAPTR include abnormal phosphate regulation in neurons,gastrointestinal tract, and liver, hypocalciuria, and hypocalcemia. Thepolynucleotide sequences encoding NAPTR may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dip stick, pin, ELISA or chip assays utilizingfluids or tissues from patient biopsies to detect altered NAPTRexpression. Such qualitative or quantitative methods are well known inthe art.

In a particular aspect, the nucleotide sequences encoding NAPTR may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingNAPTR may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding NAPTR in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of NAPTR, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes NAPTR, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding NAPTR may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5'>3') and another with antisense(3'<5'), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

Methods which may also be used to quantitate the expression of NAPTRinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236).The speed of quantitation of multiple samples may be accelerated byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or calorimetricresponse gives rapid quantitation.

In another embodiment of the invention, the nucleic acid sequences whichencode NAPTR may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome or to a specificregion of the chromosome using well known techniques. Such techniquesinclude FISH, FACS, or artificial chromosome constructions, such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial PI constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

FISH (as described in Verma et al. (1988) Human Chromosomes: A Manual ofBasic Techniques, Pergamon Press, New York, N.Y.) may be correlated withother physical chromosome mapping techniques and genetic map data.Examples of genetic map data can be found in the 1994 Genome Issue ofScicnce (265:1981f). Correlation between the location of the geneencoding NAPTR on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, NAPTR, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenNAPTR and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to NAPTR large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with NAPTR, or fragments thereof, and washed. Bound NAPTR isthen detected by methods well known in the art. Purified NAPTR can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding NAPTR specificallycompete with a test compound for binding NAPTR. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with NAPTR.

In additional embodiments, the nucleotide sequences which encode NAPTRmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I BRAITUT02 cDNA Library Construction

The BRAITUT02 cDNA library was constructed from brain tumor tissue(specimen #0172; Mayo Clinic, Rochester Minn.). The pathology reportindicated that a stage IV grade 2 renal cell carcinoma was the source ofthe primary tumor which had metastasized to the brain and formed an excerebral meningeal lesion. The patient was being treated with Decadron(Dexamethasone; Merck Sharp & Dohme, West Point Pa.) for cerebraledema/hemorrhage and with Dilantin (Phynction; Parke-Davis, MorrisPlains, N.J.) for seizures.

The frozen tissue was homogenized and lysed using a BrinkmannHomogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury N.J.). Thelysate was centrifuged over a 5.7 M CsCl cushion using a Beckman SW28rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for 18hours at 25,000 rpm at ambient temperature. The RNA was extracted withphenol chloroform pH 4.0, precipitated using 0.3 M sodium acetate and2.5 volumes of ethanol, resuspended in DEPC-treated water and DNasetreated at 37° C. The RNA was re-extracted with phenol chloroform pH 4.0and precipitated using sodium acetate and ethanol as before. The RNA wasthen isolated using the Qiagen Oligotex kit (QIAGEN Inc; ChatsworthCalif.) and used to construct the cDNA library.

The RNA was handled according to the recommended protocols in theSuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat.#18248-013; Gibco/BRL Gaithersburg Md.). cDNAs were fractionated on aSepharose CL4B column (Cat. #275105, Pharmacia), and those cDNAsexceeding 400 bp were ligated into pSport I. The plasmid pSport I wassubsequently transformed into DH5a™ competent cells (Cat. #18258-012,Gibco/BRL).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL Prep96 Plasmid Kit (Catalog #26173 QIAGEN, Inc.). The recommended protocolwas employed except for the following changes: 1) the bacteria werecultured in 1 ml of sterile Terrific Broth (Catalog #22711, Gibco/BRL)with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) the cultures wereincubated for 19 hours after the wells were inoculated and then lysedwith 0.3 ml of lysis buffer; 3) following isopropanol precipitation, theplasmid DNA pellet was resuspended in 0.1 ml of distilled water. Afterthe last step in the protocol, samples were transferred to a Beckman96-well block for storage.

The cDNAs were sequenced by the method of Sanger F and AR Coulson (1975;J Mol Biol 94:441f), using a Hamilton Micro Lab 2200 (Hamilton, RenoNev.) in combination with Peltier Thermal Cyclers (PTC200 from MJResearch, Watertown Mass.) and Applied Biosystems 377 or 373 DNASequencing Systems; and the reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

Each cDNA was compared to sequences in GenBank using a search algorithmdeveloped by Applied Biosystems and incorporated into the INHERIT™ 670sequence analysis system. In this algorithm, Pattern SpecificationLanguage (TRW Inc, Los Angeles, Calif.) was used to determine regions ofhomology. The three parameters that determine how the sequencecomparisons run were window size, window offset, and error tolerance.Using a combination of these three parameters, the DNA database wassearched for sequences containing regions of homology to the querysequence, and the appropriate sequences were scored with an initialvalue. Subsequently, these homologous regions were examined using dotmatrix homology plots to distinguish regions of homology from chancematches. Smith-Waterman alignments were used to display the results ofthe homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT-670 sequence analysis system using the methods similar to thoseused in DNA sequence homologies. Pattern Specification Language andparameter windows were used to search protein databases for sequencescontaining regions of homology which were scored with an initial value.Dot-matrix homology plots were examined to distinguish regions ofsignificant homology from chance matches.

BLAST, which stands for Basic Local Alignment Search Tool (Altschul, S.F. (1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990) J. Mol. Biol.215:403-410), was used to search for local sequence alignments. BLASTproduces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs. BLAST is useful for matches which do notcontain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

An HSP consists of two sequence fragments of arbitrary but equal lengthswhose alignment is locally maximal and for which the alignment scoremeets or exceeds a threshold or cutoff score set by the user. The BLASTapproach is to look for HSPs between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. The parameter E establishes the statisticallysignificant threshold for reporting database sequence matches. E isinterpreted as the upper bound of the expected frequency of chanceoccurrence of an HSP (or set of HSPs) within the context of the entiredatabase search. Any database sequence whose match satisfies E isreported in the program output.

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. 1993 and1990, supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding NAPTR occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of NAPTR to Full Length

Incyte Clone 754412.est (SEQ ID NO:5) was used to design oligonucleotideprimers to extend the partial nucleotide sequence to full length. Oneprimer was synthesized to initiate extension in the antisense direction(XLR=CTTGATGCTCCCATGAGAAAACTGG, SEQ ID NO:6), and the other wassynthesized to extend sequence in the sense direction(XLF=AGGATTTTCGAGCATAGCACCTGTC, SEQ ID NO:7). PCR using these primersallowed the extension of the known partial sequence "outward" andproduced three amplicons which were subsequently sequenced using theshotgun method (Messing, J., Crea, R., Seeburg, P. (1981) Nucleic AcidsRes. 9:309-321).

The initial primers were designed using OLIGO® 4.06 primer analysissoftware (National Biosciences), or another appropriate program. Optimumprimers are generally 22-30 nucleotides in length, have a GC content of50% or more, and anneal to the target sequence at temperatures about68°-72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

Extension of Incyte Clone 754412.est (SEQ ID NO:5) to full length wasaccomplished in a single PCR experiment. Essential components of theexperiment included a mixture of two commercial libraries, liver andleukocytes (Gibco/BRL, Gaithersburg Md.), the XL-PCR kit (Perkin Elmer),40 pmol each of the XLF and XLR primers, and the recommendedconcentrations of all other components of the kit. PCR was performedusing the Peltier Thermal Cycler (PTC200; MJ Research, Watertown Mass.)and the following parameters:

    ______________________________________                                        Step 1       94° C. for 1 min (initial denaturation)                                 Step 2 65° C. for 1 min                                    Step 3 68° C. for 6 min                                                Step 4 94° C. for 15 sec                                               Step 5 65° C. for 1 min                                                Step 6 68° C. for 7 min                                                Step 7 Repeat step 4-6 for 15 additional cycles                               Step 8 94° C. for 15 sec                                               Step 9 65° C. for 1 min                                                Step 10 68° C. for 7:15 min                                            Step 11 Repeat step 8-10 for 12 cycles                                        Step 12 72° C. for 8 min                                               Step 13 4° C. (and holding)                                          ______________________________________                                    

After PCR, 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis in a low concentration (about 0.6-0.8%) agarose mini-gelto determine if the sequence was extended. The bands thought to containthe largest products were selected and cut out of the gel. Furtherpurification involved gel extraction using a method such as QIAQuick™(QIAGEN Inc). After the DNA was recovered, Kienow enzyme was used totrim single-stranded, nucleotide overhangs creating blunt ends tofacilitate religation and cloning.

The reaction products were precipitated in ethanol and redissolved in 13μl of ligation buffer. After 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook J et al, supra).The whole transformation mixture was incubated for one hour at 37° C.and plated on Luria Bertani (LB)-agar (Sambrook J et al, supra)containing 2× carbenicillin (2×Carb). The following day, severalcolonies were randomly picked from each plate and cultured in 150 μl ofliquid LB/2×Carb medium placed in an individual well of acommercially-available, sterile 96-well microtiter plate. The next day,5 μl of each overnight culture was transferred into a non-sterile96-well plate. After being diluted 1:10 with water, 5 μl of each samplewas transferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rtth DNA polymerase, a vector primer and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

    ______________________________________                                        Step 1     94° C. for 60 sec                                             Step 2 94° C. for 20 sec                                               Step 3 55° C. for 30 sec                                               Step 4 72° C. for 90 sec                                               Step 5 Repeat steps 2-4 for an additional 29 cycles                           Step 6 72° C. for 180 sec                                              Step 7 4° C. (and holding)                                           ______________________________________                                    

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the full length sequence of 745412 (SEQ ID NO:2) can beused to obtain 5' sequences, promoters or regulatory elements fromappropriate genomic libraries.

VI Labeling and Use of Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³² P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN®, Boston, Mass.).The labeled oligonucleotides are substantially purified with SephadexG-25 superfine resin column (Pharmacia & Upjohn). A portion containing10⁷ counts per minute of each of the sense and antisenseoligonucleotides is used in a typical membrane based hybridizationanalysis of human genomic DNA digested with one of the followingendonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II; DuPontNEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

VII Antisense Molecules

Antisense molecules to the NAPTR-encoding sequence, or any part thereof,is used to inhibit in vivo or in vitro expression of naturally occurringNAPTR. Although use of antisense oligonucleotides, comprising about 20base-pairs, is specifically described, essentially the same procedure isused with larger cDNA fragments. An oligonucleotide based on the codingsequences of NAPTR, as shown in FIG. 1, is used to inhibit expression ofnaturally occurring NAPTR. The complementary oligonucleotide is designedfrom the most unique 5' sequence as shown in FIG. 1 and used either toinhibit transcription by preventing promoter binding to the upstreamnontranslated sequence or translation of an NAPTR-encoding transcript bypreventing the ribosome from binding. Using an appropriate portion ofthe signal and 5' sequence of SEQ ID NO:2, an effective antisenseoligonucleotide includes any 15-20 nucleotides spanning the region whichtranslates into the signal or 5' coding sequence of the polypeptide asshown in FIG. 1.

VIII Expression of NAPTR

Expression of NAPTR is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, pSport, previously used for thegeneration of the cDNA library is used to express NAPTR in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion ofNAPTR into the bacterial growth media which can be used directly in thefollowing assay for activity.

IX Demonstration of NAPTR Activity

NAPTR can be assayed by injecting Xenopus laevis oocytes at stages V andVI with NAPTR mRNA (10 ng per oocyte) and incubating for 3 days at 18°C. in OR2 medium (82.5 mM NaCl, 2.5 mM KCL, 1 mM CaCl₂, 1 mM MgCl2, 1 mMNa₂ HPO₄, 5 mM Hepes, 3.8 mM NaOH, 50 ug/ml gentamycin, pH 7.8) beforeswitching to standard uptake medium (100 mM NaCl, 2 mM KCL, 1 mM CaCl₂,1 mM MgCl2, 10 mMHepes/Tris pH 7.5). Uptake of phosphate is initiated byadding 0.1 mMKH₂ PO₄ containing 30 uCi of ³² P in uptake medium andincubating for 30 minutes. Uptake is terminated by washing the oocytesthree times in Na⁺ -free medium, measuring the incorporated ³² P, andcomparing with controls (Ni, B. (1993) supra).

X Production of NAPTR Specific Antibodies

NAPTR that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

XI Purification of Naturally Occurring NAPTR Using Specific Antibodies

Naturally occurring or recombinant NAPTR is substantially purified byimmunoaffinity chromatography using antibodies specific for NAPTR. Animmunoaffinity column is constructed by covalently coupling NAPTRantibody to an activated chromatographic resin, such as CnBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing NAPTR is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof NAPTR (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/NAPTR binding (eg, a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and NAPTR iscollected.

XII Identification of Molecules Which Interact with NAPTR

NAPTR or biologically active fragments thereof are labeled with ¹²⁵ IBolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled NAPTR, washed and any wells withlabeled NAPTR complex are assayed. Data obtained using differentconcentrations of NAPTR are used to calculate values for the number,affinity, and association of NAPTR with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 7                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: BRAITUT02                                                        (B) CLONE: 754412                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  Met Gln Val Asp Glu Thr Leu Ile Pro Arg - #Lys Val Pro Ser Leu        Cys                                                                               1               5 - #                 10 - #                 15             - -  Ser Ala Arg Tyr Gly Ile Ala Leu Val Leu - #His Phe Cys Asn Phe Thr                   20     - #             25     - #             30                  - -  Thr Ile Ala Gln Asn Val Ile Met Asn Ile - #Thr Met Val Ala Met Val               35         - #         40         - #         45                      - -  Asn Ser Thr Ser Pro Gln Ser Gln Leu Asn - #Asp Ser Ser Glu Val Leu           50             - #     55             - #     60                          - -  Pro Val Asp Ser Phe Gly Gly Leu Ser Lys - #Ala Pro Lys Ser Leu Pro       65                 - # 70                 - # 75                 - # 80       - -  Ala Lys Ser Ser Ile Leu Gly Gly Gln Phe - #Ala Ile Trp Glu Arg Trp                       85 - #                 90 - #                 95              - -  Gly Pro Pro Gln Glu Arg Ser Arg Leu Cys - #Ser Ile Ala Leu Ser Gly                   100     - #            105     - #            110                 - -  Met Leu Leu Gly Cys Phe Thr Ala Ile Leu - #Ile Gly Gly Phe Ile Ser               115         - #        120         - #        125                     - -  Glu Thr Leu Gly Trp Pro Phe Val Phe Tyr - #Ile Phe Gly Gly Val Gly           130             - #    135             - #    140                         - -  Cys Val Cys Cys Leu Leu Trp Phe Val Val - #Ile Tyr Asp Asp Pro Val       145                 - #150                 - #155                 -         #160                                                                             - -  Ser Tyr Pro Trp Ile Ser Thr Ser Glu Lys - #Glu Tyr Ile Ile Ser        Ser                                                                                              165 - #                170 - #                175            - -  Leu Lys Gln Gln Val Gly Ser Ser Lys Gln - #Pro Leu Pro Ile Lys Ala                   180     - #            185     - #            190                 - -  Met Leu Arg Ser Leu Pro Ile Trp Ser Ile - #Cys Leu Gly Cys Phe Ser               195         - #        200         - #        205                     - -  His Gln Trp Leu Val Ser Thr Met Val Val - #Tyr Ile Pro Thr Tyr Ile           210             - #    215             - #    220                         - -  Ser Ser Val Tyr His Val Asn Ile Arg Asp - #Asn Gly Leu Leu Ser Ala       225                 - #230                 - #235                 -         #240                                                                             - -  Leu Pro Phe Ile Val Ala Trp Val Ile Gly - #Met Val Gly Gly Tyr        Leu                                                                                              245 - #                250 - #                255            - -  Ala Asp Phe Leu Leu Thr Lys Lys Phe Arg - #Leu Ile Thr Val Arg Lys                   260     - #            265     - #            270                 - -  Ile Ala Thr Ile Leu Gly Ser Leu Pro Ser - #Ser Ala Leu Ile Val Ser               275         - #        280         - #        285                     - -  Leu Pro Tyr Leu Asn Ser Gly Tyr Ile Thr - #Ala Thr Ala Leu Leu Thr           290             - #    295             - #    300                         - -  Leu Ser Cys Gly Leu Ser Thr Leu Cys Gln - #Ser Gly Ile Tyr Ile Asn       305                 - #310                 - #315                 -         #320                                                                             - -  Val Leu Asp Ile Ala Pro Arg Tyr Ser Ser - #Phe Leu Met Gly Ala        Ser                                                                                              325 - #                330 - #                335            - -  Arg Gly Phe Ser Ser Ile Ala Pro Val Ile - #Val Pro Thr Val Ser Gly                   340     - #            345     - #            350                 - -  Phe Leu Leu Ser Gln Asp Pro Glu Phe Gly - #Trp Arg Asn Val Phe Phe               355         - #        360         - #        365                     - -  Leu Leu Phe Ala Val Asn Leu Leu Gly Leu - #Leu Phe Tyr Leu Ile Phe           370             - #    375             - #    380                         - -  Gly Glu Ala Asp Val Gln Glu Trp Ala Lys - #Glu Arg Lys Leu Thr Arg       385                 - #390                 - #395                 -         #400                                                                             - -  Leu                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1643 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: BRAITUT02                                                        (B) CLONE: 754412                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -  AGAACGGTGA GGATGACCGA CGTATAGGCG AGAGCCTAGG TACGCCATGC - #CAGGTCAC    CG    60                                                                        - -  GTCCGGCAAT TCCCGGGTCG ACCCACGCGT CCGCTTGGAG GGACGCTGGG - #TTCAACTTG    A   120                                                                         - -  AGCCCTTCCA CAGACATTAA GTCGGTGAAA ACCATTCACT AGGAGAGGAG - #AAACACAAT    G   180                                                                         - -  GCCACCAAGA CAGAGTTGAG TCCCACAGCA AGGGAGAGCA AGAACGCACA - #AGATATGCA    A   240                                                                         - -  GTGGATGAGA CACTGATCCC CAGGAAAGTT CCAAGTTTAT GTTCTGCTCG - #CTATGGAAT    A   300                                                                         - -  GCCCTCGTCT TACATTTCTG CAATTTCACA ACGATAGCAC AAAATGTCAT - #CATGAACAT    C   360                                                                         - -  ACCATGGTAG CCATGGTCAA CAGCACAAGC CCTCAATCCC AGCTCAATGA - #TTCCTCTGA    G   420                                                                         - -  GTGCTGCCTG TTGACTCATT TGGTGGCCTA AGTAAAGCCC CAAAGAGTCT - #TCCTGCAAA    G   480                                                                         - -  TCCTCAATAC TTGGGGGTCA GTTTGCAATT TGGGAAAGGT GGGGCCCTCC - #ACAAGAACG    A   540                                                                         - -  AGCAGACTCT GCAGCATTGC TTTATCAGGA ATGTTACTGG GATGCTTTAC - #TGCCATCCT    C   600                                                                         - -  ATAGGTGGCT TCATTAGTGA AACCCTTGGG TGGCCCTTTG TCTTCTATAT - #CTTTGGAGG    T   660                                                                         - -  GTTGGCTGTG TCTGCTGCCT TCTCTGGTTT GTTGTGATTT ATGATGACCC - #CGTTTCCTA    T   720                                                                         - -  CCATGGATAA GCACCTCAGA AAAAGAATAC ATCATATCCT CCTTGAAACA - #ACAGGTCGG    G   780                                                                         - -  TCTTCTAAGC AGCCTCTTCC CATCAAAGCT ATGCTCAGAT CTCTACCCAT - #TTGGTCCAT    A   840                                                                         - -  TGTTTAGGCT GTTTCAGCCA TCAATGGTTA GTTAGCACAA TGGTTGTATA - #CATACCAAC    T   900                                                                         - -  TACATCAGCT CTGTGTACCA TGTTAACATC AGAGACAATG GACTTCTATC - #TGCCCTTCC    T   960                                                                         - -  TTTATTGTTG CCTGGGTCAT AGGCATGGTG GGAGGCTATC TGGCAGATTT - #CCTTCTAAC    C  1020                                                                         - -  AAAAAGTTTA GACTCATCAC TGTGAGGAAA ATTGCCACAA TTTTAGGAAG - #TCTCCCCTC    T  1080                                                                         - -  TCAGCACTCA TTGTGTCTCT GCCTTACCTC AATTCCGGCT ATATCACAGC - #AACTGCCTT    G  1140                                                                         - -  CTGACGCTCT CTTGCGGATT AAGCACATTG TGTCAGTCAG GGATTTATAT - #CAATGTCTT    A  1200                                                                         - -  GATATTGCTC CAAGGTATTC CAGTTTTCTC ATGGGAGCAT CAAGAGGATT - #TTCGAGCAT    A  1260                                                                         - -  GCACCTGTCA TTGTACCCAC TGTCAGCGGA TTTCTTCTTA GTCAGGACCC - #TGAGTTTGG    G  1320                                                                         - -  TGGAGGAATG TCTTCTTCTT GCTGTTTGCC GTTAACCTGT TAGGACTACT - #CTTCTACCT    C  1380                                                                         - -  ATATTTGGAG AAGCAGATGT CCAAGAATGG GCTAAAGAGA GAAAACTCAC - #TCGTTTATG    A  1440                                                                         - -  AGTTATCCCA CCTTGGATGG AAAAGTCATT AGGCACCGTA TTGCATAAAA - #TAGAAGGCT    T  1500                                                                         - -  CCGTGATGAA AATACCAGTG AAAAGATTTT TTTTTCCTGT GGCTCTTTTC - #AATTATGAG    A  1560                                                                         - -  TCAGTTCATT ATTTTATTCA GACTTTTTTT TGAGAGAAAT GTAAGATGAA - #TAAAAATTC    A  1620                                                                         - -  AATAAAATGA TAACTAAGAA TGC          - #                  - #                  1643                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 467 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 450532                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -  Met Gln Met Asp Asn Arg Leu Pro Pro Lys - #Lys Val Pro Gly Phe Cys        1               5 - #                 10 - #                 15              - -  Ser Phe Arg Tyr Gly Leu Ser Phe Leu Val - #His Cys Cys Asn Val Ile                   20     - #             25     - #             30                  - -  Ile Thr Ala Gln Arg Ala Cys Leu Asn Leu - #Thr Met Val Val Met Val               35         - #         40         - #         45                      - -  Asn Ser Thr Asp Pro His Gly Leu Pro Asn - #Thr Ser Thr Lys Lys Leu           50             - #     55             - #     60                          - -  Leu Asp Asn Ile Lys Asn Pro Met Tyr Asn - #Trp Ser Pro Asp Ile Gln       65                 - # 70                 - # 75                 - # 80       - -  Gly Ile Ile Leu Ser Ser Thr Ser Tyr Gly - #Val Ile Ile Ile Gln Val                       85 - #                 90 - #                 95              - -  Pro Val Gly Tyr Phe Ser Gly Ile Tyr Ser - #Thr Lys Lys Met Ile Gly                   100     - #            105     - #            110                 - -  Phe Ala Leu Cys Leu Ser Ser Val Leu Ser - #Leu Leu Ile Pro Pro Ala               115         - #        120         - #        125                     - -  Ala Gly Ile Gly Val Ala Trp Val Val Val - #Cys Arg Ala Val Gln Gly           130             - #    135             - #    140                         - -  Ala Ala Gln Gly Ile Val Ala Thr Ala Gln - #Phe Glu Ile Tyr Val Lys       145                 - #150                 - #155                 -         #160                                                                             - -  Trp Ala Pro Pro Leu Glu Arg Gly Arg Leu - #Thr Ser Met Ser Thr        Ser                                                                                              165 - #                170 - #                175            - -  Gly Phe Leu Leu Gly Pro Phe Ile Val Leu - #Leu Val Thr Gly Val Ile                   180     - #            185     - #            190                 - -  Cys Glu Ser Leu Gly Trp Pro Met Val Phe - #Tyr Ile Phe Gly Ala Cys               195         - #        200         - #        205                     - -  Gly Cys Ala Val Cys Leu Leu Trp Phe Val - #Leu Phe Tyr Asp Asp Pro           210             - #    215             - #    220                         - -  Lys Asp His Pro Cys Ile Ser Ile Ser Glu - #Lys Glu Tyr Ile Thr Ser       225                 - #230                 - #235                 -         #240                                                                             - -  Ser Leu Val Gln Gln Val Ser Ser Ser Arg - #Gln Ser Leu Pro Ile        Lys                                                                                              245 - #                250 - #                255            - -  Ala Ile Leu Lys Ser Leu Pro Val Trp Ala - #Ile Ser Ile Gly Ser Phe                   260     - #            265     - #            270                 - -  Thr Phe Phe Trp Ser His Asn Ile Met Thr - #Leu Tyr Thr Pro Met Phe               275         - #        280         - #        285                     - -  Ile Asn Ser Met Leu His Val Asn Ile Lys - #Glu Asn Gly Phe Leu Ser           290             - #    295             - #    300                         - -  Ser Leu Pro Tyr Leu Phe Ala Trp Ile Cys - #Gly Asn Leu Ala Gly Gln       305                 - #310                 - #315                 -         #320                                                                             - -  Leu Ser Asp Phe Phe Leu Thr Arg Asn Ile - #Leu Ser Val Ile Ala        Val                                                                                              325 - #                330 - #                335            - -  Arg Lys Leu Phe Thr Ala Ala Gly Phe Leu - #Leu Pro Ala Ile Phe Gly                   340     - #            345     - #            350                 - -  Val Cys Leu Pro Tyr Leu Ser Ser Thr Phe - #Tyr Ser Ile Val Ile Phe               355         - #        360         - #        365                     - -  Leu Ile Leu Ala Gly Ala Thr Gly Ser Phe - #Cys Leu Gly Gly Val Phe           370             - #    375             - #    380                         - -  Ile Asn Gly Leu Asp Ile Ala Pro Arg Tyr - #Phe Gly Phe Ile Lys Ala       385                 - #390                 - #395                 -         #400                                                                             - -  Cys Ser Thr Leu Thr Gly Met Ile Gly Gly - #Leu Ile Ala Ser Thr        Leu                                                                                              405 - #                410 - #                415            - -  Thr Gly Leu Ile Leu Lys Gln Asp Pro Glu - #Ser Ala Trp Phe Lys Thr                   420     - #            425     - #            430                 - -  Phe Ile Leu Met Ala Ala Ile Asn Val Thr - #Gly Leu Ile Phe Tyr Leu               435         - #        440         - #        445                     - -  Ile Val Ala Thr Ala Glu Ile Gln Asp Trp - #Ala Lys Glu Lys Gln His           450             - #    455             - #    460                         - -  Thr Arg Leu                                                              465                                                                           - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 560 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 507415                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -  Met Glu Phe Arg Gln Glu Glu Phe Arg Lys - #Leu Ala Gly Arg Ala Leu        1               5 - #                 10 - #                 15              - -  Gly Arg Leu His Arg Leu Leu Glu Lys Arg - #Gln Glu Gly Ala Glu Thr                   20     - #             25     - #             30                  - -  Leu Glu Leu Ser Ala Asp Gly Arg Pro Val - #Thr Thr His Thr Arg Asp               35         - #         40         - #         45                      - -  Pro Pro Val Val Asp Cys Thr Cys Phe Gly - #Leu Pro Arg Arg Tyr Ile           50             - #     55             - #     60                          - -  Ile Ala Ile Met Ser Gly Leu Gly Phe Cys - #Ile Ser Phe Gly Ile Arg       65                 - # 70                 - # 75                 - # 80       - -  Cys Asn Leu Gly Val Ala Ile Val Ser Met - #Val Asn Asn Ser Thr Thr                       85 - #                 90 - #                 95              - -  His Arg Gly Gly His Val Val Val Gln Lys - #Ala Gln Phe Asn Trp Asp                   100     - #            105     - #            110                 - -  Pro Glu Thr Val Gly Leu Ile His Gly Ser - #Phe Phe Trp Gly Tyr Ile               115         - #        120         - #        125                     - -  Val Thr Gln Ile Pro Gly Gly Phe Ile Cys - #Gln Lys Phe Ala Ala Asn           130             - #    135             - #    140                         - -  Arg Val Phe Gly Phe Ala Ile Val Ala Thr - #Ser Thr Leu Asn Met Leu       145                 - #150                 - #155                 -         #160                                                                             - -  Ile Pro Ser Ala Ala Arg Val His Tyr Gly - #Cys Val Ile Phe Val        Arg                                                                                              165 - #                170 - #                175            - -  Ile Leu Gln Gly Leu Val Glu Gly Val Thr - #Tyr Pro Ala Cys His Gly                   180     - #            185     - #            190                 - -  Ile Trp Ser Lys Trp Ala Pro Pro Leu Glu - #Arg Ser Arg Leu Ala Thr               195         - #        200         - #        205                     - -  Thr Ala Phe Cys Gly Ser Tyr Ala Gly Ala - #Val Val Ala Met Pro Leu           210             - #    215             - #    220                         - -  Ala Gly Val Leu Val Gln Tyr Ser Gly Trp - #Ser Ser Val Phe Tyr Val       225                 - #230                 - #235                 -         #240                                                                             - -  Tyr Gly Ser Phe Gly Ile Phe Trp Tyr Leu - #Phe Trp Leu Leu Val        Ser                                                                                              245 - #                250 - #                255            - -  Tyr Glu Ser Pro Ala Leu His Pro Ser Ile - #Ser Glu Glu Glu Arg Lys                   260     - #            265     - #            270                 - -  Tyr Ile Glu Asp Ala Ile Gly Glu Ser Ala - #Lys Leu Met Asn Pro Val               275         - #        280         - #        285                     - -  Thr Lys Phe Asn Thr Pro Trp Arg Arg Phe - #Phe Thr Ser Met Pro Val           290             - #    295             - #    300                         - -  Tyr Ala Ile Ile Val Ala Asn Phe Cys Arg - #Ser Trp Thr Phe Tyr Leu       305                 - #310                 - #315                 -         #320                                                                             - -  Leu Leu Ile Ser Gln Pro Ala Tyr Phe Glu - #Glu Val Phe Gly Phe        Glu                                                                                              325 - #                330 - #                335            - -  Ile Ser Lys Val Gly Leu Val Ser Ala Leu - #Pro His Leu Val Met Thr                   340     - #            345     - #            350                 - -  Ile Ile Val Pro Ile Gly Gly Gln Ile Ala - #Asp Phe Leu Arg Ser Arg               355         - #        360         - #        365                     - -  His Ile Met Ser Thr Thr Asn Val Arg Lys - #Leu Met Asn Cys Gly Gly           370             - #    375             - #    380                         - -  Phe Gly Met Glu Ala Thr Leu Leu Leu Val - #Val Gly Tyr Ser His Ser       385                 - #390                 - #395                 -         #400                                                                             - -  Lys Gly Val Ala Ile Ser Phe Leu Val Leu - #Ala Val Gly Phe Ser        Gly                                                                                              405 - #                410 - #                415            - -  Phe Ala Ile Ser Gly Phe Asn Val Asn His - #Leu Asp Ile Ala Pro Arg                   420     - #            425     - #            430                 - -  Tyr Ala Ser Ile Leu Met Gly Ile Ser Asn - #Gly Val Gly Thr Leu Ser               435         - #        440         - #        445                     - -  Gly Met Val Cys Pro Ile Ile Val Gly Ala - #Met Thr Lys His Lys Thr           450             - #    455             - #    460                         - -  Arg Glu Glu Trp Gln Tyr Val Phe Leu Ile - #Ala Ser Leu Val His Tyr       465                 - #470                 - #475                 -         #480                                                                             - -  Gly Gly Val Ile Phe Tyr Gly Val Phe Ala - #Ser Gly Glu Lys Gln        Pro                                                                                              485 - #                490 - #                495            - -  Trp Ala Glu Pro Glu Glu Met Ser Glu Glu - #Lys Cys Gly Phe Val Gly                   500     - #            505     - #            510                 - -  His Asp Gln Leu Ala Gly Ser Asp Glu Ser - #Glu Met Glu Asp Glu Val               515         - #        520         - #        525                     - -  Glu Pro Pro Gly Ala Pro Pro Ala Pro Pro - #Pro Ser Tyr Gly Ala Thr           530             - #    535             - #    540                         - -  His Ser Thr Val Gln Pro Pro Arg Pro Pro - #Pro Pro Val Arg Asp Tyr       545                 - #550                 - #555                 -         #560                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 272 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: BRAITUT02                                                        (B) CLONE: 754412                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - -  ATTTATATCA ATGTCTTAGA TATTGCTCCA AGGTATTCCA GTTTTCTCAT - #GGGAGCAT    CA    60                                                                        - -  AGAGGATTTT CGAGCATAGC ACCTGTCATT GTACCCACTG TCAGTGGATT - #TCTTCTTAG    T   120                                                                         - -  CAGGACCCTG AGTTTGGGTG GAGGAATGTC TTCTTCTTGC TGTTTGCCGT - #TAACCTGTT    A   180                                                                         - -  GGACTACTCT TCTACCTCAT ATTTGGAGAA GCAGATGTCC AAGAATGGGC - #TAAAGAGAG    A   240                                                                         - -  AAACTCACTC GTTTATGAAG TTATCCCACC TT      - #                  - #             272                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: XLR                                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - -  CTTGATGCTC CCATGAGAAA ACTGG         - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: XLF                                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - -  AGGATTTTCG AGCATAGCAC CTGTC         - #                  - #                   25                                                                    __________________________________________________________________________

What is claimed is:
 1. An isolated and purified polynucleotide fragmentencoding a polypeptide comprising the amino acid sequence of SEQ IDNo:1.
 2. A hybridization probe comprising the polynucleotide fragment ofclaim
 1. 3. An isolated and purified polynucleotide fragment comprisingSEQ ID No:2.
 4. An isolated and purified polynucleotide fragment whichis completely complementary to the polynucleotide of claim
 1. 5. Ahybridization probe comprising the polynucleotide fragment of claim 4.6. An expression vector comprising the polynucleotide of claim
 1. 7. Ahost cell containing the expression vector of claim
 6. 8. A method forproducing a polypeptide comprising the amino acid sequence of SEQ IDNO:1, the method comprising the steps of:a) culturing the host cell ofclaim 7 under conditions suitable for the expression of the polypeptide;and b) recovering the polypeptide from the host cell culture.
 9. Amethod for detection of a polynucleotide encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 in a biological samplecontaining nucleic acid material, the method comprising the steps of:a)hybridizing the polynucleotide of claim 4 to the nucleic acid materialof the biological sample, thereby forming a hybridization complex; andb) detecting the hybridization complex, wherein the presence of thehybridization complex correlates with the presence of the polynucleotideencoding the polypeptide in the biological sample.